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Keysight Technologies
Single Molecule Force Spectroscopy (SMFS)
Using the 7500AFM
Application Note

Introduction Avidin-Biotin Interaction Study
Using SMFS
The atomic force microscope (AFM) 1 is
Single Molecule Force Spectroscopy can
well known for its high imaging resolution
be used to study the interaction of pro-
even at ambient environment and room
teins. For this, an upgraded sensor design
temperature. Besides that, it can be used to
is necessary. One binding partner has to be
measure forces, accurately. Its force resolu-
bound to the apex of the AFM tip using a
tion at room temperature is high enough to
lexible linker 9, 10, 11, while the other bind-
measure forces as small as weak bindings
ing partner is immobilized on the sample
between biological molecules 2, 3, 4, 5, 6. Those
surface. Interaction force measurements
abilities make the AFM, especially force dis-
are monitored by performing force distance
tance based AFM methods a valuable tool to
cycles. Hereby, the tip is approached until it
investigate biological functions 7, 8.
touches the surface. Eventually this results
in a bond forming between the ligand (e.g.
For biological processes weak interactions
an organic molecule, peptide, toxin of full
play a crucial role. Usually, ligand-receptor
protein) bound to the tip and the receptor
type complexes of proteins (e.g. antibodies,
molecule (typically a protein) immobilized
enzymes) and/or other organic biomolecules
on the sample surface. Subsequent retrac-
(e.g. vitamins, hormones) are formed. The
tion results in loading of the bond with an
lifetime and stability of such formed com-
increasing force ramp. At a certain force the
plexes is optimized for its biological func-
bond ruptures and the bent cantilever jumps
tion. Therefore measuring that weak interac-
off the surface into its equilibrium posi-
tion of proteins is important to understand
tion. The force at which the bond breaks is
its function.
termed "rupture force".
Here, we show how the Keysight Tech-
The breaking of the bond is driven by ther-
nologies, Inc. 7500 AFM can be utilized to
mal luctuations rather than by purely me-
improve the molecular understanding of
chanical dissociation 12. In fact, the loading
complex biological systems. In particular
of the bond results in lowering the energy
we show how interactions between proteins
barrier, which leads to a shorter lifetime
can be studied by Single Molecule Force
of the interaction, i.e. faster loading yields
Spectroscopy (SMFS), yielding the energy
larger rupture forces. Therefore, rupture
landscape.
forces depend on the applied force load-
02 | Keysight | Single Molecule Force Spectroscopy (SMFS) Using the 7500AFM - Application Note

ing rate and the details of the functional
relation of bond lifetime and applied force.
A detailed theoretical consideration yields
a direct link between such single molecule
pulling experiments and bulk experiments.
But the single molecule approach gives
access to the full spectrum of individual
properties of the ligand-receptor pair in-
stead of an averaged value gained by bulk
experiments 13.

In this paper we show how the energy
landscape of the avidin-biotin interac-
tion can be determined. Avidin is a protein
made of four identical subunits (homo-
tetramer), where each can bind to biotin
with a high degree of afinity and speci-
icity. It is produced in oviducts of birds,
reptiles and amphibians and deposited in
the whites of their eggs. In chicken white
egg it makes up approximately 0.05% of
Figure 1. Force distance cycles showing avidin-biotin unbinding events. Avidin-biotin interaction is indicated
the total protein content. The biological by negative forces and a characteristic non-linear stretching of the tether (~30-60 nm). An unbinding event is
function of avidin is not exactly known, characterized by a sudden increase of the force due to a rupture of the bond (~65 nm). The rupture force (force
although it has been postulated that it is a at which unbinding occurs) is depending on the force loading rate. The higher the loading rate (900, 3600 and
18000 pN/s) the higher the rupture forces.
bacterial growth inhibitor, by binding biotin
which bacteria need. Biotin, also known
as vitamin H, is a water soluble B-vitamin
(B7) and is necessary for cell growth, the
production of fatty acids, and the metabo-
lism of fats and amino acids.

Avidin and biotin bind with a dissociation
constant of roughly 10 -15 M, making it one
of the strongest non-covalent bonds in
nature. In fact, the afinity is so high that it
is extremely resistant to any type of dena-
turing agent. This stability has prompted
its use for in situ attachment of labels in
a broad variety of applications, including
immunoassays (like ELISA), DNA hybrid-
ization and localization of antigens in cells
and tissues.

Here, the avidin-biotin interaction was
investigated using single molecule force
spectroscopy. For this, biotin was cova-
Figure 2. Distribution of rupture forces as a function of the applied loading rate. Applying higher force loading
lently bound to the AFM tip and avidin rates result in a shift of rupture forces to larger values.
was immobilized to a freshly cleaved mica
sheet. Single molecule interactions were
monitored by acquiring force distance tion of many of such unbinding events assuming that for unbinding, an energy
cycles using the above prepared tip and (>100) yielded a distribution of rupture barrier needs to be crossed in a thermally
sample. Interaction between avidin and forces with a mean value of 50 pN and activated regime 12.
biotin was represented as a character- a standard deviation of 19pN at a force
istic nonlinear force signal, arising from loading rate of 900 pN/s. The nature of this Another consequence of the stochastic
the elastic extension of the distensible distribution are not measurement errors nature is that the unbinding forces depend
PEG linker stretching, allowing for distin- of consecutive measurements, in fact it on the time scale of the measurements.
guishing speciic unbinding events from is the stochastic nature of the unbinding The most probable unbinding force is
unspeciic adhesion (Figure 1). Evalua- process, itself. It can be understood by shifted to higher values for larger force
03 | Keysight | Single Molecule Force Spectroscopy (SMFS) Using the 7500AFM - Application Note

loading rates (Figure 1 and Figure 2). In
fact there is a linear dependence of the
most probable unbinding force and the
logarithm of the loading rate (Figure 3).
From this dependence the separation of
the energy barrier from the equilibrium
position x and the kinetic off-rate con-
stant koff can be determined 12. Therefore
the unbinding forces were measured at
different pulling speeds, ranging from
300nm s-1 to 24000nm s-1. The force load-
ing rate was calculated from the retrac-
tion velocity times the effective spring
constant, which is the slope of the force
distance curve at the point of rupture.
From the most probably unbinding force
versus force loading rate dependence (Fig-
ure 3) x and koff were determined to be
0.16

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